PROJECT SUMMARY Type 2 diabetes (T2D) is driven by progressive pancreatic islet dysfunction. Despite this central role, the mechanisms underlying this dysfunction in human islets are not well understood. Using a holistic approach that allows for study of both the pancreas and isolated islets from the same donor, our lab is characterizing molecular signatures from recent-onset (<7 years, on oral hypoglycemics) T2D individuals compared to non- diabetic controls. We discovered that isolated islets in recent-onset T2D have significant dysfunction with reduced insulin secretion and elevated glucagon secretion in response to various secretagogues. By immunocytochemistry and RNA-sequencing, the T2D ? and ? cells have reduced levels of key islet-enriched transcription factors (TFs), PAX6 and NKX2.2. Studies from mice have shown that these TFs are crucial for maintaining cell identity and function of adult ? cells; however, little is known about their role in human ? or ? cells. Additionally, we have found that T2D islets show evidence of increased inflammatory signaling as well as increased numbers of intraislet macrophages. Thus, this proposal seeks to investigate the mechanisms of TF loss and inflammation in T2D islet dysfunction. We hypothesize that PAX6 and NKX2.2 are required for normal human ? and ? cell function and identity and are altered in the inflammatory environment of type 2 diabetes. To test this hypothesis, we will first examine the role of PAX6 and NKX2.2 in maintenance of human ? and ? cell phenotype and function. Using virally delivered shRNA, we will knock down each TF in human ? and ? cells and measure islet function through insulin and glucagon secretion in response to secretagogues and phenotype through gene expression analysis. We will also perform rescue experiments to see if expressing PAX6 and NKX2.2 in T2D islets can restore cell function and phenotype. These results will determine whether PAX6 and/or NKX2.2 are necessary in human ? and ? cells and will define their role in T2D. Secondly, we will determine the role of the T2D inflammatory environment in PAX6 and NKX2.2 reduction. We will test this by culturing normal islets in cytokine cocktails reflective of either local or systemic signals and measuring expression of PAX6 and NKX2.2. We will also test if transplantation into an immunodeficient environment of T2D islets with and without macrophages and endothelial cells (local sources of inflammatory signals) will result in recovery of PAX6 and/or NKX2.2. Finally, we will characterize these intraislet macrophages and endothelial cells to define their contribution to the T2D inflammatory environment. This aim will determine if inflammation leads to reversible PAX6 or NKX2.2 reduction and provide insights into the cause of islet dysfunction in T2D. Overall, this proposal will help define mechanisms behind pancreatic islet dysfunction in T2D and aid in the development of targeted therapeutics.